Affiliations: 1) Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases,NIH, Bethesda,MD; 2) Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases,NIH, Bethesda,MD; 3) Center for Cancer Research, National Cancer Institute, NIH, Bethesda,MD
Keywords: f. insulators/boundary elements; c. remodeling complexes
Chromatin insulators are DNA-protein complexes that promote chromatin 3D organization by mediating interactions between distant genomic sites. Insulators can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. Altering insulator function can lead to defects in cellular differentiation and organismal development. In Drosophila, the well-studied gypsy insulator consists of three core proteins: Su(Hw), Mod(mdg4)67.2, and CP190. Multimerization of insulator proteins forms insulator bodies, of which normal localization is correlated with proper insulator function. To identify factors required for insulator body formation, we used a cell line expressing a functional Mod(mdg4)67.2-GFP fusion protein and performed a high-throughput visual RNAi screen. This strategy identified Nurf301, a nucleosome remodeling factor, as a potential novel regulator of gypsy insulator body formation.
Previous work showed that nucleosomes are shifted at Su(Hw) sites in Nurf301 null hemocytes, but it remained unclear how Nurf301 mechanistically affects gypsy insulator function. First, we found that Nurf301 promotes gypsy-dependent insulator barrier activity in a ubiquitous manner using an in vivo luciferase assay. Co-IP and IP-mass spec results indicated that Nurf301 physically interacts with CP190, Su(Hw), and Mod(mdg4)67.2. Furthermore, Nurf301 co-localizes with insulator proteins, and depletion of Nurf301 extensively alters the distribution of gypsy insulator proteins on chromatin. ChIP-seq and NeuRNA-seq profiles in cells lacking Nurf301 and Su(Hw) revealed that Nurf301 and Su(Hw) mutually affect the binding of one another on chromatin and regulate expression of a similar set of genes. Correlation between altered nucleosome positioning and reducedSu(Hw) binding in Nurf301 null cells suggested a functional relationship. Analysis of chromatin accessibility using ATAC-seq supported the hypothesis that Su(Hw) serves as a docking site for recruitment of Nurf301, and subsequently, CP190. Finally, DNA Oligopaint FISH demonstrated that nuclear compaction of gypsy binding sites is specifically reduced with concomitant loss of CP190 binding after depletion of Nurf301. By providing evidence of mutual functional interaction between Nurf301 and Su(Hw), our data provide new insights into how a nucleosome remodeling factor interacts with insulator proteins to regulate the nuclear organization and gene regulation.